Spacer acquisition during the type III-A CRISPR-Cas immune response

项目摘要 CRISPR-CAS系统为细菌和古菌提供针对它们的病毒(噬菌体)的适应性免疫。 CRISPR-Cas免疫反应的特点是获得了一种感染记忆，其形式为 来自入侵的噬菌体基因组的一小段DNA序列。这个序列被称为“间隔区”，被整合到 然后转录并加工成CRISPR RNA(CrRNA)指南。 CRISPR相关(Cas)效应器使用crRNA识别入侵噬菌体的核酸 通过碱基对互补，触发不同的防御策略。对于类型III CRISPR系统， 普遍存在于人类病原体金黄色葡萄球菌中，靶标识别导致休眠 被感染的细胞，阻止病毒复制和传播的事件。在这里，我们建议调查一项 关于这一机制的核心但尚未得到回答的问题是：如果有间隔体在 宿主，它们在获得后是如何在细菌群落中维持的？我们的中心假设是 噬菌体DNA的降解最终从宿主中消除了病毒基因组，使其能够生长 以及种群中间隔区的固定。为了调查这一点，我们将从几个方面探讨 Csm6介导的防御所需的反应是由与晚期匹配的III型CRISPR间隔区介导的 表达了病毒基因。首先，我们将定义休眠细胞是否最终死亡或能够退出这种状态， 在感染中存活并继续生长。其次，我们将确定匹配的间隔区是否晚期表达 噬菌体基因可以为携带噬菌体基因的细胞提供选择性优势，即使它们触发宿主时也是如此 休眠。第三，我们将确定这些间隔区是否真的是在感染期间获得的。在这一切中 我们将通过使用缺乏表达的突变型葡萄球菌来验证我们的中心假设 几个核酸酶，以确定它们是否需要休眠触发间隔物的固定。最后，我们 将使用突变的转座子文库，以无偏见的方式研究宿主基因对 可能与从休眠中解脱有关。 我们提出的实验旨在了解来自休眠诱导CRISPR系统的间隔物 都是固定在宿主人口中的，将填补我们在理解这一标志方面的基本知识空白 CRISPR免疫的特点：感染记忆的产生。此外，通过直接寻址 葡萄球菌噬菌体防御的基本机制，我们的建议可以促进噬菌体的成功 葡萄球菌病的治疗方法。以更间接的方式，对 III型CRISPR系统的分子机制可能导致改变这些免疫系统的用途 用于基因编辑，特别是用于开发治疗遗传病的基因疗法。

Project Summary CRISPR-Cas systems provide bacteria and archaea with adaptive immunity against their viruses (phages). The hallmark of the CRISPR-Cas immune response is the acquisition of a “memory” of infection in the form of a short DNA sequence from the invading phage genome. This sequence, known as “spacer”, is integrated into the CRISPR locus of the host and then transcribed and processed into a CRISPR RNA (crRNA) guide. CRISPR-associated (Cas) effectors use the crRNA to recognize the nucleic acids of the invading phage through base-pair complementarity and trigger different defense strategies. For Type III CRISPR systems, commonly present in the human pathogen Staphylococcus aureus, target recognition leads to the dormancy of the infected cell, an event that prevents viral replication and propagation. Here, we propose to investigate a central, yet unanswered, question about this mechanism: if there are spacers that trigger a growth arrest in the host, how are they maintained in the bacterial community after they are acquired? Our central hypothesis is that the degradation of the phage DNA eventually eliminates the viral genome from the host, enabling growth and the fixation of the spacer in the population. To investigate this, we will explore several aspects of the Csm6-mediated response required for the defense mediated by type III CRISPR spacers that match late- expressed viral genes. First, we will define whether dormant cells eventually die or are able to exit this state, survive infection and continue growing. Second, we will determine whether spacers that match late-expressed phage genes can provide a selective advantage to the cell that harbors them, even when they trigger host dormancy. Third, we will determine if these spacers are actually acquired during infection. In all these experiments we will test our central hypothesis by using mutant staphylococci lacking in the expression of several nucleases to determine if they are required for the fixation of dormancy-triggering spacers. Finally, we will use a transposon library of mutants to investigate, in an unbiased manner, the impact of host genes that could be involved in the exit from dormancy. Our proposed experiments, aimed at understanding how spacers from dormancy-inducing CRISPR systems are fixed in the host population, will fill in a fundamental knowledge gap in our understanding of the hallmark feature of CRISPR immunity: the generation of a memory of infection. In addition, by directly addressing a fundamental mechanism of phage defense of staphylococci, our proposal can facilitate the success of phage therapies for the treatment of staphylococcal disease. In a more indirect manner, the characterization of the molecular mechanisms of type III CRISPR systems can lead to avenues to repurpose these immune systems for gene editing, particularly for the development of gene therapies to treat genetic diseases.